With rare exceptions, the treatment for acute myeloid leukemia (AML) has changed little in the last 40 years and long-term overall survival has remained stable at approximately 30%. The group directed by the applicant's mentor, Dr Carl June, is recognized as a world leader in the field of genetically-engineered T cell therapy for hematologic malignancy, with well- publicized recent success in treating CD19+ B-cell leukemias. In part, the success of this therapy depends on the restricted tissue expression of CD19 and on the ability of patients to tolerate prolonged depletion of normal B cells. In contrast to B-cell leukemias, AML presents a unique set of challenges, as it is fundamentally a cancer of hematopoietic stem/progenitor cells. Therefore, the underlying challenge limiting the application of powerful immunotherapy approaches to AML is the lack of truly leukemia-specific antigens. The long-term goal of this research is to apply the CAR-T cell approach to AML. The overall objective as the next step in pursuit of the above goal is to identify safe ways to treat AML without endangering normal tissue. The central hypothesis is that careful selection of target-specific CAR constructs, either singly or in combination, can maximize on-target efficacy while minimizing the consequences of off-target toxicity. Guided by strong preliminary data, this hypothesis will be tested by pursuing three specific aims: 1) design of split-signaling CAR constructs wherein activation is conditional upon the T cell encountering two antigens, thus increasing the specificity of CAR stimulation; 2) demonstration that the myeloablative off-tumor effect of CAR-T cells can be harnessed as a novel cellular conditioning regimen for HCT; and 3) identification of novel cell-surface targets in AML using proteomics followed by bioinformatics analysis. The approach is innovative, in the applicant's opinion, as it departs from the status quo by extending both the range of available AML targets using novel approaches to target discovery, as well as the utility of existing AML targets using conditional expressing CAR constructs. The proposed research is significant, because it will contribute depth (of clinical responses) and breadth (of eligibility for potentially curative therapy) to the therapeutic arsenal against AML. Ultimately, such knowledge has the potential to vertically advance the burgeoning field of chimeric antigen receptor-redirected T cell immunotherapy in AML and other malignancies. The proposed research activities are crucial to the development of the applicant as an independently-funded physician-scientist with a focus on cellular immunotherapy. Dr Gill will receive further training in molecular biology from his mentor Dr Carl June, and training in proteomics and in bioinformatics from experienced collaborators at the University of Pennsylvania. Therefore at the conclusion of the training period, the applicant will have acquired a unique set of intellectual and technical skills that will allow him to attack the problem of AML- specific immunotherapeutics from several angles at once. In addition, this award will support a unique training experience in translational research and will establish an academic path- way for the discovery and development of new chimeric antigen receptor T cell approaches.